D2 Dopamine Receptors Expressed by Striatal Cholinergic Neurons Contribute to Action Sequence Learning One of our major aims is to understand how circuitry within the basal ganglia contributes to the control and learning of actions. We are also very interested in how neurotransmitters such as acetylcholine (ACh) and dopamine contribute to striatal circuit function and behavior. Previous studies from the Costa laboratory at LIN showed that striatal neurons encode different aspects of instrumental lever pressing in a self-paced task in which mice are required to respond 8 times (FR8) for a food reward (Jin and Costa, Nature 2010, Jin et al., Nature Neuroscience 2014). Striatal medium spiny projection neurons (MSNs) show several lever-press-related firing patterns. Some respond at the beginning or end of the 8 lever-press sequences, others fire before each press, and another subset fire throughout the sequence. This latter firing pattern appears to develop as animals become more proficient in the sequencing task. The striatal cholinergic interneurons (CINs) constitute only a small subset of the total neurons in this brain region, but have important modulatory effects on MSN firing and roles in striatal-based behaviors. These actions arise from ACh release leading to activation of both muscarinic and nicotinic-type ACh receptors. These neurons express D2-type dopamine receptors that have two important physiological functions. Activation of these receptors hyperpolarizes CINs and produces a pause in the normal tonic firing pattern exhibited by these neurons. There is evidence from a few in vivo recordings that CINs show pauses in firing during FR8 lever-pressing sequences (Jin et al., 2014). The D2 receptors on CINs also contribute to induction of long-term synaptic depression (LTD) at cortical synapses onto striatal MSNs, by inhibiting ACh release that normally suppresses key intracellular mechanisms involved in LTD induction. This form of synaptic plasticity has been implicated in striatal-based learning. Given the intriguing cellular and synaptic roles of D2 receptors on CINs, it was important to determine how these receptors might contribute to sequence learning that involves striatum. To achieve this aim, we used a Cre x lox breeding scheme with Choline Acetyltransferase (ChAT)-Cre mice. Two subtypes were used, a bacterial artificial chromosome (BAC)-Cre mouse, and an internal ribosomal entry (IRES)-Cre mouse. These mice were bred with a mouse carrying a floxed allele of a key exon of the D2 receptor to achieve knockout of the receptor in CINs (CIN-D2KO). We demonstrated the efficacy of this breeding scheme for producing the desired knockout by showing loss the D2-mediated pause of CIN tonic firing, as well as induction of afferent stimulation-induced LTD. Furthermore, this breeding scheme did not result in loss of D2 receptors on MSNs or dopaminergic neurons themselves. Mice lacking this receptor and littermate controls were trained on the self-paced FR8 lever-pressing task using a stringent criterion where sequences were re-set if animals attempted to retrieve the sucrose reward prior to completing 8 presses. This resulted in rapid learning of sequences with low variability in control mice. In contrast, CIN-D2KO mice showed slower acquisition at all phases of training, fewer presses per sequence, lower efficiency in obtaining reward, and fewer rewards earned per session. The CIN-D2KO mice also made fewer lever presses and lower breakpoint in a progressive ratio instrumental task in comparison to controls. These results were obtained regardless of which ChAT-Cre mouse was used for breeding, indicating that effects were not related to higher ACh levels known to be present in the BAC-Cre mice. The CIN-D2KO mice performed comparably to controls in other tasks, including the accelerating rotarod skill task, movement in a novel open field environment, and a lever-pressing reversal task on an FR1 schedule. Thus, the impairments were most obvious on tasks that required sustained performance driven by reward. In vivo electrophysiological recordings from putative MSNs in control mice essentially replicated previous findings from the Costa laboratory, showing the aforementioned three press-related firing patterns and the training-related increase in sustained firing of a subset of neurons throughout the sequence. In the CIN-D2KO mice, the percentage of neurons that showed any press-related firing was slightly lower than in controls. The largest difference in in the CIN-D2KO mice was the near absence of sustained-firing neurons even after extensive training. This result indicates that removal of D2 receptors from CINs impairs the ability of MSNs to encode the full sequence duration, which could contribute to poor sequence performance. To determine if this impairment involved D2 receptors on CINs in dorsal striatum (the region examined by the previous Costa laboratory studies and our in vivo recordings), we injected a Cre-dependent viral construct containing the D2 sequence into dorsal striatum of the CIN-D2KO mice. This viral expression restored accurate lever-press sequencing behavior, but interestingly did not fully rescue the deficits in the progressive ratio task. Injection of the virus into ventral striatum did not rescue any of the deficits. These findings indicate that loss of D2 in the dorsal striatum is indeed involved in the sequence task deficit. The data also provide evidence that the behavioral impairment is not due to a developmental change in striatal function, but rather reflects an ongoing problem when D2 receptors are not present in the adult striatum. Altogether, our findings indicate that D2 receptors expressed by CINs in dorsal striatum have important roles in sequence learning and maintaining instrumental performance under conditions of high task demand. The normal role of this receptors is most likely to reduce the influence of ACh release on MSNs during crucial phases of task learning and performance. The fact that we observed deficits in the CIN-D2KO mice early in training before sequences are optimal in control mice, and the observation that gradual development of sustained firing of MSNs is lost in these knockout mice suggest a role for the receptor in plasticity that helps to shape learning. However, further work is needed to determine if the loss of CIN pausing, the loss of LTD, or both underlie these behavioral deficits.